ABSTRACT The potential of oncolytic virotherapy as a cancer treatment has been clearly demonstrated in clinical trials, however the overall efficacies of currently available oncolytic viruses is modest. It has become clear that a balance between the oncolytic phase and immune phase of this treatment modality needs to be reached in order to maximize the potential of cure. Mengovirus, a picornaviral pathogen, represents an improved class of oncolytic viruses because of its ability to undergo preclinical evaluation in immunocompetent animals including mice and primates. Mengovirus can cause encephalitis and myocarditis, however, we enhanced its safety by truncating the polycytidine tract (a pathogenic determinant) and by incorporating neuron and cardiac specific microRNAs into the viral genome. Treatment with this dual-detargeted virus, vMC24-NC, results in complete regression of syngeneic mouse multiple myeloma plasmacytomas. However, metastatic recurrence was observed in 50% of the treated mice indicating a need for improving the immune phase of this virotherapy. The goal of this proposal is to develop a Mengovirus therapy expressing immunostimulatory proteins in order to boost the immune phase during the oncolytic phase. Incorporation of foreign genes into picornaviral genomes is inhibited by their limited insertion capacity. Therefore we developed a Mengovirus-based replicon where the genes encoding the capsid proteins were replaced with a secreted luciferase gene. This replicon results in foreign gene expression, genome replication, and encapsidation with short-term spread when co- administered with vMC24-NC. We hypothesize that we can improve the overall spread of the replicon construct by creating a co-dependence system with an additional helper replicon encoding the capsid proteins and/or optimizing the treatment protocol based on dose, ratio, and sequence of replicon and helper/virus administration. These studies will produce for the first time a single-shot system where a potent and safe oncolytic picornavirus can be armed with immunostimulatory proteins and tested in immunocompetent animals resulting in a superior oncolytic virotherapy. With this in mind, we propose the following specific aims: Aim 1. Compare the efficiency of spread and genetic stability of our capsid-substituted (vector) replicons in cells coinfected with a) replication-competent vMC24-NC or b) capsid-encoding vMC24-NC derived (helper) replicons that are co-dependent with the vector replicon for nonstructural protein functions. Aim 2. Determine how the efficiency of intratumoral vector genome expression and spread is impacted by (i) relative dose of vector and helper (ii) temporal sequencing of vector and helper. Aim 3. Evaluate the therapeutic efficacy of combination therapies using immunostimulatory Mengovirus replicons with vMC24-NC or helper replicons.